The present invention in general falls within the field of devices for the immunologic detection of antigens.
More specifically, the invention relates to a planar support having an ultraflat surface useful for detecting antigens by label-free techniques, more particularly by AFM (Atomic Force Microscopy) imaging. The planar support of the invention is preferably provided in the form of an array of antibodies, such as for example a nanoarray or microarray of antibodies.
In the state of the art, the main analytical assay for detecting proteins of biological interest, such as for instance proteins useful as biomarkers, is represented by the ELISA (Enzyme-Linked ImmunoSorbent Assay) technique, which is known to be characterized by a detection limit of about 5 pg/ml and requires a minimum sample volume of about 50 μl.
Recently, the ELISA technique has been in part substituted with the protein microarray technique, which exhibits the advantage of being able to use smaller sample volumes and allowing for the simultaneous analysis of many analytes. Among which, antibody microarrays are known. A microarray of antibodies includes multiple antibody spots arranged in an ordered way, wherein each spot has a micrometric size and consists of multiple molecules of a single capture antibody directed against a predetermined individual antigen. With such a technique, the antigens are captured in a specific way by the antibodies and are mainly detected by fluorescent labeling techniques. The detection limit of the conventional microarrays is within the pM-fM range, the minimum volume of sample is in the order of picoliters, the size of the spots is typically comprised in the range of 100-300 μm in diameter and the quantity of the different antibodies/arrays is less than 500.
In this context, a further miniaturization would certainly be advantageous as it would allow to obtain a higher density in the array, use smaller volumes and reduce the amount of analyte. Several approaches were attempted to develop the so-called ultramicroarrays (Nettikadan S et al. Mol Cell Proteomics 2006, 5:895-901; Lee K B et al. Science 2002, 295:1702-1705; Sekula S et al. Small 2008, 4:1785-1793; Zhang G J et al. J Nanosci Nanotechnol 2007, 7:410-417). Although technologies for printing a plurality of identical spots of nanometric size are already available (nanoprinting, nanolithography, nanodispensing), the issue of functionalizing each spot with a different antibody has not been completely solved yet (Wingren C et al. Drug Discov Today 2007, 12:813-819). Furthermore, the currently available scanners that are used for fluorescence detection are not capable of resolving spots of nanometric size (resolution≧1 μm) and the usefulness of the label-free techniques for detecting single molecules in disease proteomics has not been demonstrated yet.
In order to overcome the previously stated problems, particularly as regards the functionalizing of arrays with a plurality of different antibodies and the detection of nanometric spots, the present inventors concentrated their efforts on the creation of a planar support having an ultraflat surface, such that the support is suitable for manufacturing a nanoarray (or optionally a microarray) capable of being detected by AFM imaging. Indeed, such a technique allows for the detection of the formation of an Ab-Ag immunocomplex at concentrations that are lower than the fM range, without the need of using chemical labeling techniques such as for example the fluorescent labeling.
AFM imaging is known to allow for the detection of single molecules, under the proviso that the substrate is extremely smooth. For this reason, the substrate functionalizing method is particularly critical in AFM imaging.
Several functionalizing methods are known which allow the capture antibodies to maintain their own orientation and function. The most common approach consists in using antibody-binding proteins, such as protein A and protein G, which are able to specifically recognize the Fc region of antibodies. Even though such a method is simple and inexpensive, as it does not require the modification of the capture antibodies, it has the disadvantage of not being suitable to be used with the AFM imaging technique, because of the high molecular size of protein A and protein G. Other approaches described in the state of the art appear to be costly, both in terms of money and time, requiring the use of tagged recombinant antibodies (Steinhauer C et al. Proteomics 2006, 6:4227-4234; Torrance L et al. J Virol Methods 2006, 134:164-170; Vallina-Garcia R et al. Biosens Bioelectron 2007, 23:210-217).
In the light of the above, a device for detecting antigens, such as for instance a nanoarray or microarray of capture antibodies, which is characterized by a high sensitivity, which allows for the use of very small sample volumes and relies on label-free detection techniques would be extremely desirable.